WO2012136909A2 - Thixotropic α-lactalbumin hydrogels, method for preparing same and uses thereof - Google Patents

Abstract

The present invention relates to shear-thinning α-lactalbumin hydrogels, which have a threshold and are thixotropic, to a method for preparing same and to the use thereof.

Description

 Thixotropic hydrogels based on α-lactalbumin, process for their preparation and their uses

 The present invention relates to the preparation of hydrogels having advantageous properties and using a by-product of the dairy industry: Γα-lactalbumin, the hydrogels thus obtained and their use, in particular for the preparation of biomaterials.

 The hydrogels produced in the context of the present invention have particular rheological characteristics which make them interesting for certain applications; indeed, it is about thixotropic gels able, under a sufficient stress, to deconstruct to a liquid state and then to restructure once left at rest.

 The protein used to produce the hydrogels is Γα-lactalbumin (hereinafter referred to as -α-lactalbumin), in particular bovine α-La. This milk protein is found more particularly in whey or whey. It therefore has the advantage of being a natural protein and available in large quantities in the cheese industry, the manufacture of cheese being made from the "curd" milk (caseins precipitated and cleared of whey).

 A gel is composed of a molecule that, in the presence of an appropriate solvent, self-assembles via chemical or physical interactions, and organizes itself into a network. Specific methods leading to gel formation depend on the physicochemical properties and interactions of the gel components. When the solvent is water we speak of hydrogel.

A hydrogel is a network of polymer chains initially soluble in water but which have become insoluble after crosslinking. Hydrogels are super-absorbent natural or synthetic polymers (they can contain more than 99% water). They also have a degree of flexibility very similar to that of natural fabrics, because of their high water content. Their physical characteristics, permeability and biocompatibility make them excellent candidates for biomaterials used in medical applications including delivery of molecules as well as tissue engineering. Depending on the nature of the cross-links, the hydrogels are separated into two categories: chemical gels derived from traditional cross-linking methods with covalent bonds and physical gels resulting from self-assembly of macromolecules (by carbohydrate or protein) and constitute networks held together by molecular entanglements and / or weak bonds (hydrophobic, ionic bonds, hydrogen bridges and van der Waals forces); the Molecular interactions of these physical gels can be disrupted by environmental changes such as temperature, pH, ionic strength, light or even a given biological molecule. Gelation is therefore reversible in response to one or more of these stimuli.

 The preparation of hydrogels from milk proteins is already known.

Milk proteins are natural vehicles for bioactive molecules thanks to their structural and physicochemical properties such as their ability to bind ions and small molecules or their self-assembly and gelation properties. Despite the amount of milk protein-based materials (caseins or whey proteins) already used in a variety of industries, many studies on these proteins are continuing and are aimed at developing new and innovative nano-objects: such as edible and biodegradable food films. made from milk proteins combined with other natural biopolymers (Chen 1995, Le Tien, Vachon et al., 2001); coacervates or nano-vesicles of caseins or other milk proteins that have been developed especially as delivery systems (Audic, Chaufer et al., 2003, Semo, Kesselman et al., 2007, Livney 2010); or milk protein gels.

 Among the milk protein gels, mention may first be made of the gelling of caseins which can be obtained under different conditions: by the acidity at pH 4.6; by the action of rennet whose active ingredient is chymosin, an enzyme that effects proteolytic cleavage resulting in the aggregation of micelles, and which is used for the encapsulation of probiotic bacteria (Heidebach, Forst et al., 2009a); by the action of transglutaminase, an enzyme that bridges certain amino acids with each other (essentially glutamic acid and lysine), which contributes to the polymerization of proteins and is also used for the microencapsulation of probiotic cells (Heidebach, Forst et al., 2009b); by the action of genipin, a natural protein crosslinking agent, for the controlled delivery of molecules at the intestinal level (contraction of the hydrogel at acidic pH (stomach) then swelling at neutral pH and thus release of the active ingredient (intestine) ) (Song, Zhang et al., 2009).

It is also known to induce the gelling of whey proteins by raising the temperature (Paulsson, Hegg et al., 1986). Like other hydrogels, gels made from whey proteins exhibit a pH-sensitive swelling behavior that can be used for targeted delivery of molecules (Gunasekaran, Ko et al., 2007). The β-lactoglobulin gels, also induced by temperature, are highly studied and have been characterized using various techniques such as TEM (transmission electron microscopy), Wide angle X-ray scattering (WAXS), and Fourier transform infrared spectroscopy (FTIR) (Kavanagh, Clark et al., 2000). The denaturation of proteins can lead to hydrophobic interactions between them, especially β-lactoglobulin (b-Lg) and bovine serum albumin (BSA) which have free thiols and can therefore be interchanged disulfide bridges: for example, BSA nanospheres have been made with a magnetic particle and / or a photo-sensitizer and can be used in cancer treatments (Rodrigues, Simioni et al., 2009). A cold gelation process has recently been developed and makes the gels thus obtained potentially interesting for the delivery of molecules. The advantage of these gels lies in the fact that the heat-sensitive bioactives can be added after the heat treatment of the whey but before the gelling which is then induced by the addition of salts (preferably calcium) or by the decrease of the pH.

 Numerous rheometry measurements were performed on β-lactoglobulin gels alone (Gosal, Clark et al 2004b) or in admixture with α-lactalbumin (Kavanagh, Clark et al., 2000). They made it possible to determine the factors, pH and ionic strength, affecting the physical characteristics of the gels (Loveday, Rao et al., 2009). The β-lactoglobulin (b-Lg) gels induced by prolonged exposure to high temperatures (80 ° C) form in two stages and consist mainly of partially degraded β-lactoglobulin polypeptides (Akkermans, Venema et al., 2008). Oboroceanu, Wang et al., 2010). The b-Lg gels can also be obtained by dissolving the protein in a particular water / alcohol mixture. The rheological and structural characteristics of these gels are different (Gosal, Clark et al 2004a, Gosal, Clark and others 2004c, Loveday, Rao and others 2009).

 Other known types of gels made from milk proteins are formed from nanotubes of α-lactalbumin (Ipsen, Otte et al., 2001). These nanotubes are formed by self-assembly of α-lactalbumin fragments and under certain conditions (minimum protein concentration and protein / calcium ratio) (Graveland-Bikker, Ipsen et al., 2004, Ipsen and Otte 2007). The peculiarity of these fibers is that they are obtained only in the presence of calcium and with a protein which has previously undergone proteolysis prepared by a particular serine protease, extracted from Bacillus licheniformis (Ipsen and Otte 2007).

The formation of gels from intact α-lactalbumin, and induced by high temperatures (80 ° C), has been shown at neutral pH in two articles studying in particular the influence of α-lactalbumin on the gelation of β- lactoglobulin (Hines and Foegeding 1993; Kavanagh, Clark et al. 2000). In particular, the team of Kavanagh et al. followed the gelling at 80 ° C of different concentration ratios between b-Lg and a-La as well as single proteins constituting their controls. The control corresponding to the only at pH 7 has a gelling time 10 times slower than that observed with the b-Lg alone. These a-La gels did not attract the attention of researchers and were therefore not characterized from a structural and rheological point of view. An α-La gel prepared according to the conditions described by Kavanagh et al. has been prepared by the Applicant (see Example 3); it has macroscopic characteristics different from those of the hydrogels developed by the Applicant; it is hard, elastic and irreversible (it does not change shape when it is stressed).

 Finally, C. Blanchet's thesis ("Protein folding and formation of amyloid fibers, the case of Γα-lactalbumin", supported on 23/06/2008) describes a process for the preparation of α-lactalbumin suspension at 40 °. C, at pH 2 at different salt concentrations (NaCl from 0 to 150 mM), these methods are however not implemented for the specific purpose of preparing gels but to characterize the behavior of Pa-lactalbumin.

 The gels obtained with 150 mM NaCl have, however, been characterized, they have a rheofluidifying behavior; it is not suggested in this thesis that such described gels may exhibit a thixotropic behavior and such behavior can not be deduced from the tests presented in this thesis.

 The Applicant has reproduced the process for preparing α-lactalbumin hydrogel as described in this thesis and was able to confirm experimentally that the gels thus obtained are not thixotropic (see Example 4 below).

 A shear thinning (or pseudoplastic) fluid is a fluid whose viscosity decreases if the shear stress or the rate of deformation applied to it increases (see the preamble of Example 2).

 It should be noted that shear thinning fluids are not necessarily thixotropic, for example carbopol gels (Piau 2007, Tokpavi, Jay et al., 2009).

 In the course of its work, the Applicant has succeeded in developing rheofluidifying, thixotropic and thixotropic α-lactalbumin hydrogels.

The commonly accepted definition of thixotropy (Mewis 1979, Pignon, Magnin et al., 1998) is as follows: a material is commonly called thixotropic, if from a state of rest for a sufficiently long period, its Viscosity decreases with time and its structure changes, when a constant shear gradient is applied to it. In a reversible way, if the shear is interrupted, the viscosity increases again, the material gradually covers the consistency and the structure that it had at rest (Figure 1).

 This characteristic is very much in demand especially for the spreading and the application of numerous food products (milk protein or soy protein gels WO2008 / 130252), in paints or cosmetics. It can also be very useful as a non-invasive method of in situ injection of hydrogels into targeted delivery of molecules or for tissue reconstruction. An article in Nature Nanotechnology shows that thixotropic gels composed for example of PEG-silica can be used and have various advantages to 3D cell culture (Pek, Wan et al., 2008).

The hydrogels according to the invention are so-called threshold as a minimum stress to be applied to allow the material to flow beyond a deformation designated critical strain y _c mice, as part of the present invention, a value greater than 0.1, preferably between 0.1 and 1. The critical strain is reached when a sufficiently high stress (threshold stress) is applied to a hydrogel completely at rest so that said hydrogel begins to flow; it is identified graphically by crossing the curve of the storage module G 'and that of the loss module G "(see example 2 and Figure 6).

More particularly, the present invention relates to a method for preparing a hydrogel of a-lactalbumin from an aqueous suspension? -Lactalbumin at a concentration C _a- between 5 and 60 mg / mL, comprising the following steps:

 a) suspending Γα-lactalbumin in an acidic aqueous solution having an ionic strength of less than or equal to 60 mM, preferably less than 50 mM and still more preferably having a value of 30 mM; said suspending consisting of:

 (a1) preparing an acidic aqueous solution having a proton concentration expressed in mM as determined by the sum: (numeric value of Ca-expressed in g / L) + 10;

(a2) suspending α-lactalbumin in said acidic aqueous solution; and (a3) if necessary, adjusting the pH to a value between 1.5 and 2.5, preferably between 1.8 and 2.2, and even more preferably, the pH is 2.0;

 b) forming the gel from said suspension of α-lactalbumin obtained at the end of step a); said formation of the gel is carried out under the following conditions:

 at a temperature below 60 ° C., preferably between 35 and

55 ° C;

 under agitation having an intensity defined by a Reynolds number between 37 and 1000, preferably between 300 and 500;

 - for 10 hours to 1 week (168 hours), in particular, between 48 and 96 hours, and

 in the absence of evaporation of water of said suspension of α-lactalbumin. The aqueous suspension of α-lactalbumin may also be referred to as solution in the following.

 Surprisingly, despite a low or no ionic strength of the aqueous solution used for the preparation of the α-La hydrogel, the Applicant has found that it is still possible to obtain a thixotropic hydrogel and that this hydrogel exhibited satisfactory stability over time; indeed, the Applicant has observed that low ionic strength hydrogels are more stable in time than those obtained with high ionic strength (> 60 mM) which become viscous with time. In addition, the hydrogel preparation having a very low salt content is advantageous for example for uses as food texturizer.

 Α-Lactalbumin is a small 14 kDa globular metalloprotein with 4 disulfide bridges, is structurally homologous to lysozyme and requires the presence of calcium for a functional structure. It is the most abundant protein of human whey and the second protein after b-Lg in bovine whey. It is part of the model proteins in refolding studies because it is able under specific conditions to adopt a particular partially folded state: the "molten globule" (MG) state (Permyakov and Berliner 2000).

Α-La is the least allergenic milk protein (Restani, Ballabio et al., 2009) and despite some resistance to digestive enzymes in the presence of other milk proteins, the peptides released by the action of different proteases on the skin. 'a-La are widely studied. Many processes for whey enrichment in α-La or for purification of α-La are used on an industrial scale: filtration on membranes; column chromatography (IEX, SEC, HIC); Enzymatic hydrolysis to degrade caseins or b-Lg, combined with membrane filtration or isoelectric precipitation combined with heat treatment to precipitate a-La (Kamau, Cheison et al., 2010).

 The process according to the invention can be carried out with any α-lactalbumin; according to a particular mode of implementation, it is a-lactalbumin from the cheese industry from cow's milk, sheep, goat, buffalo, camel, mare, ...

 In the context of the implementation of the process according to the invention, the α-lactalbumin source used preferably has a purity of at least 85%, preferably at least 90%. It is also possible to implement the process according to the invention with lactoserum enriched in α-lactalbumin and having a content of at least 45% by weight of α-lactalbumin.

 Preferably, the α-lactalbumin suspension obtained at the end of step a) is filtered. This filtration can be carried out with a filter having a cutoff threshold such that it passes objects having a molecular weight less than or equal to 20 kDa, such as a-La proteins, but retains microorganisms and other contaminants; it is thus possible to use cutoff threshold filters less than or equal to 0.5 μηι, for example commercial filters at 0.22 μη.

 The implementation of the gel formation step b) is carried out with a homogeneous temperature of the total of the α-lactalbumin suspension.

 Stirring of the α-lactalbumin slurry carried out during this step b) must also be homogeneous throughout the stirring vessel.

 In order to characterize the intensity of the stirring, which must be low but not zero, regardless of the geometry and the size of the container and the stirrer, the Applicant has determined a range of Reynolds number value adapted to the implementation of the method according to the invention; thus, the Reynolds number must be between 37 and 1000, preferably between 300 and 500. The methods for determining the Reynolds number are detailed in Example 1 below.

By way of example, when the process is carried out on a laboratory scale (volume of suspension of α-lactalbumin of between 1 ml and 100 ml), the stirring can be carried out using a rotary plate or else with a magnetic bar whose length is between 70 and 90% of the diameter of said container; in these cases, the stirring speed is between 10 and 300 revolutions per minute (rpm).

 The α-Gels are formed at acidic pH, favorable to the MG form and thus to the fibrillation of the protein. The pH value is extremely important for the formation of gels since it is impossible for example at pH 3 (Kavanagh, Clark et al., 2000).

 During the implementation of step (a1) of the process according to the invention, the proton content making it possible to obtain the acidic pH necessary for the formation of the hydrogel can be obtained with a strong acid, for example HC1.

 It is essential to carry out the acidification according to step (a1) before the step

(a2) suspending the a-La.

The ionic strength used for the preparation of the hydrogel according to the invention and which is between 0 and 60 mM is obtained by the optional addition of salt which can be chosen from alkali metal or alkaline earth metal halides, such as by example NaCl, KCl, MgCl ₂ , CaCl ₂ ...; alkali metal or alkaline earth metal carbonates or mixtures thereof; phosphates, such as, for example, sodium or potassium phosphate or sulphates such as, for example, sodium or magnesium sulphate.

 The salt concentration of the aqueous solution of α-La is conventionally determined by those skilled in the art depending on the desired ionic strength.

 According to a particular variant of implementation of the method according to the invention, it is implemented without the addition of salt.

 Example 2 which follows shows the rheological characterization of the α-La hydrogels obtained by the process according to the invention.

 The present invention also relates to α-lactalbumin hydrogels that can be obtained according to the method of the invention.

 It is more specifically hydrogels having an α-lactalbumin content of between 5 and 60 mg / mL; a pH of between 1.5 and 2.5, preferably between 1.8 and 2.2 and more preferably, the pH is 2.0; an ionic strength of less than or equal to 60 mM, preferably less than 50 mM and, still more preferably, having a value of 30 mM.

These hydrogels are characterized by a rheofluidifying fluid behavior, stress threshold (a flow occurs when a critical strain between 0.1 and 1 is applied) and thixotropic; the viscosity of these hydrogels decreases if the shear stress or the rate of deformation applied to them increases, moreover, these hydrogels are destructured under the influence of shear (liquefaction) and restructure when shearing ceases (solidification).

 Products from natural sources such as milk or whey, α-lactalbumin hydrogels according to the invention are part of sustainable development. The product is natural and therefore biodegradable, biocompatible, non-toxic and considered GRAS (generally recognized as safe). In addition, the production process is compatible with green chemistry processes, that is to say gentle chemistry that preserves the environment since it uses neither solvent nor crosslinking agent.

 The milk proteins, and in particular Ρα-lactalbumin, have remarkable properties:

 their physico-chemical properties are well known: structure, stability, solubility, affinity for metals, hydrophobicity ...

 - Γ self-assembly of proteins is in the form of fibers, one of the foundations of nanotechnology;

 - they have high nutritional values;

 they present various biological effects observed mainly from peptides derived from their proteolysis (Madureira, Pereira et al., 2007).

 In addition, the use of these proteins on an industrial scale is suitable because:

 - their source, whey, has a low cost;

 the processes for purifying whey proteins on an industrial scale are already known;

 - the quantities available are considerable: firstly because of the amount of a-La contained in the whey (more than 1 g / L in bovine whey) and secondly because of the available quantities of whey since by-product of the cheese industry and therefore material to be valued.

 Whey or whey is the main waste of cheese factories and caseineries. The main whey proteins are β-lactoglobulin (b-Lg), Γα-lactalbumin (a-La), immunoglobulins, bovine serum albumin (BSA) and lactoferrin (Lf).

The quantities of whey available in the world are considerable. In France, 15 billion liters of whey are produced each year by the production of cow's cheese. In the absence of a valuation solution, the whey not self-consumed by cattle is dumped in rivers or spread in fields, with adverse consequences for the natural environment: pollution of rivers, lakes and groundwater and odor nuisance. This pollution is mainly due to the fermentation of whey organic matter (lactose and nitrogenous material) and the decrease of the dissolved oxygen content of the water below an acceptable threshold. Indeed, the chemical and biological oxygen demand (COD and BOD) of this waste are high (COD 50 to 70 g / L) and make it a seriously polluting material. The establishment of economically acceptable devices, aiming to collect and valorize whey, is today mandatory to reduce these damage to the environment.

 The high nutritional, functional and biological value of the a- La justifies its interest in the food, pharmaceutical and cosmetic fields. The use of the α-La hydrogels according to the invention can be envisaged in the form of hydrogels as such, of course, but also in the form of films by spreading (spin-coating) and then drying the gels, or in the form of extrusion or electrospinning of the gels.

 The Applicant has demonstrated that the α-La hydrogels having an α-lactalbumin content of between 5 and 60 mg / ml and prepared at acidic pH, that is to say between 1.5 and 2. , 5, preferably between 1.8 and 2.2 and even more preferably at pH 2.0, regardless of their ionic strength, are rheofiuidative, threshold and thixotropic.

 Although these hydrogels have an acid pH at the end of their preparation, it is possible to increase their pH in order to adapt it to the desired uses of said hydrogels while preserving their rheological properties.

 Thus, according to another of its objects, the present invention relates to thixotropic hydrogels having an α-lactalbumin content of between 5 and 60 mg / ml and repaired at an acidic pH but whose pH could be increased thereafter , for use where their rheological properties prove advantageous.

 Consisting of edible products, the hydrogels of a-La find in particular many applications in the field of the food industry.

Thus, the present invention relates to a food product comprising a thixotropic hydrogel of α-lactalbumin having an α-lactalbumin content of between 5 and 60 mg / ml and the use of such an α-lactalbumin hydrogel. as a texturizing food agent. According to a particular embodiment, the ionic strength of said hydrogels is between 0 and 60 mM, preferably less than 50 mM and still more preferably with a value of 30 mM. Depending on the desired consistency of the food product, it may contain between 0.5 and 98% of a-lactalbumin thixotropic hydrogel by weight relative to the total weight of said food product.

 Due to the Γα-lactalbumin properties mentioned above, the hydrogels according to the invention are particularly suitable for the preparation of biomaterials. The term "biomaterials" means materials intended to be in temporary or permanent contact with different tissues, organs or fluids of a living being, for diagnostic, preventive or therapeutic purposes; biomaterials also include materials intended to be implanted in a living organism.

 Thus, the present invention also relates to thixotropic α-lactalbumin hydrogels having an α-lactalbumin content of between 5 and 60 mg / mL, for use as a product used for the treatment and / or healing of wounds; in particular, these hydrogels have an ionic strength of between 0 and 60 mM. Indeed, α-lactalbumin hydrogels meet the criteria generally considered for dressing preparation: biocompatibility and absence of cytotoxicity; preventing dehydration of the wound with the maintenance of a moist environment; protection against dust and bacteria; maintenance of gas exchanges; easy application on the wound and easy to remove after healing.

 In addition, in order to improve their effectiveness, the hydrogels may also comprise one or more active compounds such as compounds capable of promoting cutaneous cicatrization, for example by promoting epithelialization by delivering specific active molecules (eg EGF) or antimicrobial compounds.

 Thus, the present invention relates to a dressing composed of at least one a-lactalbumin thixotropic hydrogel having an α-lactalbumin content of between 5 and 60 mg / ml; according to a particular variant, these hydrogels have an ionic strength of between 0 and 60 mM, preferably less than 50 mM and, still more preferably, having a value of 30 mM. Optionally, said dressing further comprises at least one active compound such as a healing agent or an antimicrobial agent.

 According to one variant, the dressing according to the invention comprises an external film permeable to air and impervious to liquids and microorganisms; such a film may for example be composed of polyurethane.

According to another of its objects, the present invention relates to a cosmetic composition comprising at least one thixotropic hydrogel of a-lactalbumin having an α-lactalbumin content of between 5 and 60 mg / ml, and the use of at least one such α-lactalbumin thixotropic hydrogel for the preparation of a cosmetic composition, in particular, as a moisturizing agent for the skin in compositions intended for the care of the skin (gel, cream, lotion, etc.) or as surfactant (washing products, shampoo, etc.); according to a particular variant, these hydrogels have an ionic strength of between 0 and 60 mM, preferably less than 50 mM and, still more preferably, having a value of 30 mM.

 Finally, because of their thixotropic behavior, the hydrogels having an α-lactalbumin content of between 5 and 60 mg / ml are advantageously used for the preparation of paints, in particular industrial paints. Indeed, the hydrogels allow these paints to remain solid during their transport and storage thus avoiding unwanted flow while having a spread easy to use. In addition, the hydrogels can improve the stability and conservation of industrial paints that are usually used in tanks constantly agitated. By agitation performed only at the time of their use, they also make it possible to achieve energy savings.

 Thus, the present invention also relates to the use of a thixotropic α-lactalbumin hydrogel having an α-lactalbumin content of between 5 and 60 mg / ml, for the preparation of paints, in particular industrial paints, and paints comprising such a hydrogel; according to a particular variant, these hydrogels have an ionic strength of between 0 and 60 mM, preferably less than 50 mM and, still more preferably, having a value of 30 mM.

 In addition to the foregoing, the invention also comprises other arrangements which will emerge from the description which follows, which refer to examples of implementation of the present invention, and to the appended figures in which:

 figures

 Figure 1 illustrates the behavior of thixotropic fluids whose viscosity decreases with time when a constant shear gradient is applied to it, and which, reversibly, increases again if the shear is interrupted.

Figure 2 shows the follow-up of the destructuration and restructuring at 15 ° C over time of a 20-mg / ml a-La hydrogel and 30 mM NaCl in small harmonic shear deformations. Different procedures for monitoring the evolution of the elastic modules G '(full circle) and viscous G "(empty circle) in function of time and imposed deformation (cross), have been applied; the constraints applied to each step are detailed in Example 2 which follows.

FIG. 3 represents the follow-up of the restructuring at 15 ° C. over time of a 20-mg / ml a-La hydrogel and 30 mM NaCl after a large-scale destructuration at a shear rate of 10 s. ^"1 , the elastic modulus G 'is represented in a full circle and the viscous module G" in an empty circle.

FIG. 4 illustrates the end of the follow-up of the restructuring at 15 ° C. over time of a 20-mg / ml a-La hydrogel and 30 mM NaCl after a large-scale deformation destructure at a shear rate gradient. 10 s ^"1 (step 6), then the step 7 of harmonic shear destructuring with increasing deformation amplitude, and finally the step 8 of monitoring the recovery of consistency in small deformation

Figure 5 is a comparison of the restructuring kinetics at 15 ° C time of an α-La hydrogel at 20 mg / mL and 30 mM NaCl (step 6) after large-scale destructuration at a shear rate. of 10 s ^"1 , (G 'is represented as a solid triangle and G" as an empty triangle) and (step 8) after a destructuration in small harmonic shear deformations with increasing deformation amplitude (G' is represented in a solid circle and G "in an empty circle).

Figure 6 shows the follow-up of the harmonic shear destructuration with increasing deformation amplitude at 15 ° C time of a hydrogel of a-La at 20 mg / mL and 30 mM NaCl: identification of the critical deformation y _c d about 0.2 beyond which the sol-gel transition begins to appear (step 7).

 Figure 7 shows the follow-up of the destructuration and restructuring at 15 ° C over time of a-la hydrogel in small harmonic shear deformations; 20 mg / mL - 0 mM NaCl.

 Figure 8 shows the follow-up of the destructuration and restructuring at 15 ° C over time of an a-La hydrogel in small harmonic shear deformations; 20 mg / mL - 60 mM NaCl

 Figure 9 shows the follow-up of the destructuration and restructuring at 15 ° C over time of a-La hydrogel in small harmonic shear deformations; 40 mg / mL - 60 mM NaCl.

FIG. 10 compares the levels of the viscoelastic modules G 'and G "as a function of the NaCl ionic strength and the a-La concentration: the G' modulus for a 20 mg / ml hydrogel of a-La is represented by solid disks, the G "modulus for a 20 mg / ml hydrogel of a-La is represented by empty disks, the G 'module for a The 40 mg / ml hydrogel of α-La is represented by solid triangles and the G 'modulus for a 40 mg / ml hydrogel of α-La is represented by empty triangles. The levels were compared at the same time of restructuring 500 s of step 3 corresponding to the zone of slow kinetics of restructuring.

Figure 11 is a graph that compares the critical strain levels y _c versus NaCl ionic strength and a-La concentration (empty discs at a concentration of 20 mg / ml a-La and solid discs for an a-La concentration of 40 mg / mL). For each condition, the destructuring phenomenon is very reproducible, which shows the great capacity of the system to undergo various shear stresses without undergoing any physicochemical modification or denaturation.

 Figure 12 shows the follow-up of the destructuration and restructuring at 15 ° C over time of two hydrogels of a-La at 20 mg / mL and 0 mM NaCl (one prepared from The purified, the second from whey enriched at 45% a-La) in small deformations in harmonic shear. Various procedures for monitoring the evolution of the elastic modules G '(solid circle for the hydrogel prepared from purified a-La; solid triangle for the hydrogel prepared from whey enriched with 45% a-La ) and viscous G "(empty circle for the hydrogel prepared from purified a-La, empty triangle for the hydrogel prepared from whey enriched at 45% a-La) as a function of time and imposed deformation (cross), were applied, the stresses applied during steps 1 to 3 are detailed in Example 2 which follows.

 FIG. 13 comprises two Ependorf tube images comprising, on the one hand, a hydrogel produced according to the protocol of C. Blanchet's thesis (B) and, on the other hand, a hydrogel according to the invention (A). The picture on the left shows these two hydrogels after their preparation (both are in the conical end of the tubes); the picture on the right shows these two hydrogels after stirring, the hydrogel (A) according to the invention is in the lower part of the Ependorf tube while the hydrogel (B) remained in the upper part (conical end).

 EXAMPLE 1 Determination of Reynold Number Value Range of Intensity for Agitating the Process According to the Invention

 The Reynolds number represents the intensity of the stirring; in a stirred reactor, it is equal to Re = where:

p is the density of the fluid mixed in Kg.m- ³ , V is the speed of rotation of the magnetic bar in (m / s),

 μ is the viscosity of the mixed fluid in (Pa.s) and

 d is the size of the stirring tool (for example, the length for the case of a magnetic bar) (in m).

 The speed of rotation N of the magnetic bar is defined by the speed imposed by the stirrer. For a speed range from 0 to 300 rpm.

 The relation between the speed V in m / s and the speed of rotation N in rpm is the following: V = - N

 30

 oit (X ^

 Either we can evaluate a Reynolds number: Re =

The mixed fluids (alpha-lactalbumin powder + aqueous suspension) have a viscosity very close to that of water because the concentration of alpha-lactalbumin powder is sufficiently low not to significantly modify the viscosity of the suspension during its introduction into water: consequently during the initial mixing of the suspension, the viscosity and the density of the stirred suspension will be set equal to that of the water is: p = 1000 Κ / μ ³ , μ = 10 ^{" 3} Pa.s.

The size of the magnetic bar used is d = 6x 10 ^{~ 3} m in length.

The table below lists examples of number values of

Reynolds adapted to the recommended mixture to obtain thixotropic gels from alpha-lactalbumin suspensions:

1000. 7T. (6.10- ^{3) 2}

R ^S = 30. 10-3

 EXAMPLE 2 Preparation of α-Lactalbumin Hydrogels According to the Invention

 2.1. Hydrogel prepared from the purified

The purified protein, "α-lactalbumin from bovine milk Type III, calcium depleted,>85%" sold under the reference "L6010" catalog marketed by Sigma, and freeze-dried is resuspended in an aqueous solution of HCl containing or without NaCl. The concentration of HCl depends on the final concentration of a-La. It is calculated in mM by adding 10 to the numerical value of the desired concentration of a-La.

 For example, if one wishes to place at 40 mg / ml of a-La, the concentration of HCl for resuspending will be 40 + 10 = 50 mM.

 First, the HCl solution must be prepared at the determined concentration and then NaCl added between 0 and 60 mM. Then you have to weigh the amount of a-La needed. This amount depends on the final concentration of protein and the volume of gel to be prepared. The concentrations of α-La used range from 5 to 60 mg / ml.

 The protein is dissolved in the defined volume of HCl solution and the pH is adjusted to 2.0 ± 0.1 with a few microliters of 1M HCl. The solution is placed under magnetic stirring using a magnetic bar and incubated overnight at a temperature ranging from 37 to 45 ° C. The next day is about 16 hours later, the gel is formed.

 2.2. Hydrogel prepared from whey enriched with 45% of a-La

 The foregoing protocol is reproduced using a whey enriched in the 45% by weight provided by Armor Protein Company.

 Example 3 Determination of the viscoelastic characteristics of the hydrogels of a-La by rheology

 Notions of rheology

 Rheology is a branch of physics that studies the flow or deformation of bodies under the effect of the constraints applied to them, given the speed of application of these constraints or more generally of their variation over time. .

At a high concentration of α-lactalbumin, the formation of amyloid fibers is accompanied by an increase in viscosity of the solution. When the fibers are formed, they interact with each other to form a gel. This increase in viscosity is followed by rheometry. The sample placed in a rheometer will be subjected to a certain stress (r) dependent on the shear rate (y) applied. The stress τ varies with the shear rate γ and the ratio between the two makes it possible to determine the viscosity (η) of the studied fluid. When r is proportional to /, then η is a constant and the fluid is Newtonian whereas if r is not proportional to γ, then the fluid is non-Newtonian and can be of different natures: if the viscosity η decreases when τ and / increase, then one has a shear thinning fluid;

 - Conversely, if η increases when τ and increase, then we have a rheo-thickening fluid.

 Thixotropic fluids are shear thinners, their viscosity decreases under the same stress over time due to a destructuring of the material. These fluids are reversible since when the stress is stopped, the material is restructured to recover its initial viscoelastic characteristics.

 The viscoelastic characteristics of a material are obtained by the determination of the dynamic viscosity moduli according to the Hooke law: τ = Gf where G has two components, G 'and G ", which serve to quantify the viscous or elastic behavior of the materials. G 'is the storage module (elastic) and G "is the loss module (viscous). When the elastic character dominates, G '"G" and conversely when the viscous character dominates, G' "G".

Rheometric behavior of gels

3.1. Rheometric measurements

Characterization of shear flow behavior of α-La gels was performed in rotational rheometry. The measurements were carried out using a rotary rheometer with imposed torque (ARG2, TA Instrument, 78 Guyancourt, France). The geometries used are titanium cone-plane geometries (angle 4 °, diameter 20 mm _s truncation 1 13 μηι). To avoid evaporation of the sample during measurements, the atmosphere was saturated with water around the sample. For harmonic shear measurements, a preliminary study has made it possible to define the deformation and optimum frequency levels for which the measurements belong to the linear regime domain. In this field, the imposed harmonic shear stress does not modify the rheological behavior of the suspensions, and only probes the viscoelastic modules of the gels without disturbing them. The frequency of 0.1 Hz has been defined as belonging to the linear regime irrespective of the imposed deformation and the time of restructuring of the samples. The set of measurements in harmonic shear will therefore be carried out at this frequency of 0.1 Hz. In the follow-up of the restructuring, a γ deformation of 0.01, has also been defined as not disturbing the measurement of G 'and G ", and will be systematically used to follow the restructuring of the samples. 3.2. Time tracking of destructuration - restructuring of salts under shear

3.2.1. Behavior in small deformations

 A harmonic shear consistency consistency procedure was implemented and used systematically for different samples at given concentration and ionic strength conditions.

 FIG. 2 shows a succession of harmonic shears for an α-La gel (20 mg / ml a-La - 30 mM NaCl) according to the strain amplitude conditions reported in Table I below:

 Table I: Deformation conditions applied during the procedure of destructuration- restructuring of the a-La gels in harmonic shear.

 During steps 1, 3 and 5 with a constant strain amplitude, it is possible to demonstrate the recovery of consistency of the gel corresponding to its restructuring.

During steps 2 and 4, the gradual increase in the deformation amplitude makes it possible to follow the destructuring of the gel caused by the preceding shear. At increasing amplitude of deformation, the elastic modulus G 'and viscous G "decrease steadily until a critical deformation y _c beyond which the levels fall sharply which highlights the destructuring of the gel and the passage of a behavior elastic to a viscous behavior (G 'becomes less than G ").

The resumption of consistency at the beginning of steps 3 and 5, characterized by the increase of G 'and G "over time, clearly demonstrates the thixotropic behavior of the gel. a short time restructuring Tri with a strong recovery of consistency and of the order of 300 s followed by a longer time Tr2 during which the increases of G 'and G "follow a slower kinetics. The same observations can be made by applying the first three steps described above to the hydrogel prepared from whey enriched at 45% by weight of α-La; the gel obtained is therefore also thixotropic.

3.2.2. Behavior in large deformations

 In order to demonstrate the thixotropic behavior on larger amplitude deformations, a simple shear in large deformation was imposed, followed by a harmonic shear in small deformations to follow the recovery of consistency of the gel over time. In order to highlight the effect of the shear rate on the level of destructuring achieved as well as on the kinetics of restructuring, various shear gradients in large deformation were applied.

 Table II: Deformation conditions applied during the procedures of destructuring in large deformation and restructuring follow-up of the a-La gels in harmonic shear.

Figure 3 shows the consistency recovery (step 6) after large-scale shear at a shear rate of 10 s ^-1 for 300 s, and again it can be noted that there is a first period Tri on which the elastic and viscous modules grow strongly over time, and a second period Tr2 for which a restructuring kinetics is much slower.

 Following a long restructuring follow-up over 1000 min (more than 16 h), a harmonic shear destructuration procedure with increasing strain amplitude is again applied (step 7) (see Figure 4), followed by a recovery of consistency with small deformation (step 8) (see Figure 4).

In FIG. 5, the two consistency recoveries are compared either after a simple shear in large deformation (step 6) or after a harmonic shear with increasing deformation amplitude (step 8). The results highlight different restructuring kinetics according to these two modes of destructuring used. The kinetics of increasing deformation following shear simple in large deformation is much slower than during a harmonic shear in small deformation with increasing amplitude. Indeed the shear in large deformation manages to destructure the sample to a higher level than that obtained during a shear in small deformation. This result again highlights the importance of the type of stress and its intensity on the level of destructuration reached in the sample during its shear, which is indicative of the behavior of thixotropic systems.

FIG. 6 shows the evolution of the viscoelastic modules as a function of the deformation, measured during a destructuring procedure (step 7) in harmonic shear with increasing strain amplitude. It is shown that the critical deformation y _c beyond which the gel begins to flow, identified by the crossing of G 'and G ", is of the order of 0.2.

 3.3. Effect of ionic strength on the thixotropic behavior of a-La salts

 In order to evaluate the differences in the kinetics of restructuring-destructuring of the α-La gels as well as the consistency levels achieved as a function of the ionic strength in NaCl, a procedure identical to that presented in Table I was carried out. works on various suspensions of a-La. The results are shown in Figures 7 to 9.

 Figure 10 shows the evolution of the viscoelastic modules G 'and G "as a function of the NaCl ionic strength and the concentration of α-La The levels were compared at the same time of the 500 s restructuring. stage 3 corresponding to the zone of slow kinetics of restructuring, ie on the "plateau" reached during the restructuring, it is demonstrated that the levels of G 'and G "decrease when the ionic strength increases; which corresponds to a reduction of the consistency of the gel. The increase in the concentration of α-La causes an increase in the viscoelastic modules.

In Figure 11, the destructures applied to various samples are compared; these results show that the critical strain moves to higher levels as the ionic strength increases or when the protein concentration decreases. Successive destructurations of the same sample have critical deformations of the same order of magnitude (not shown in the figure) which highlights the very good stability of the system to undergo successive stresses of destructuration-restructuring, as well as a very high good stability over time due to the reproducibility of measurements in G 'and G ". Example 4 Preparation of a q-lactalbumin hydrogel according to the conditions described in C. Blanchet's thesis

 The purpose of this test and to reproduce a suspension of α-La obtainable by the protocol described by C. Blanchet et al. then to characterize its rheological properties.

 The experiments were conducted under the conditions and according to the protocol described on page 203 of the thesis: the α-La proteins (10 mg / ml with 30 mM NaCl) are suspended, then the pH of this suspension is adjusted to 2 and the suspension is introduced into an Ependorf tube; the tube is stirred at 40 ° C. (the stirring conditions are those used for the preparation of the hydrogels according to the invention).

 In parallel, a hydrogel at 10 mg / ml of a-La with 30 mM NaCl is prepared by the method according to the invention.

 The left-hand image of FIG. 13 illustrates the appearance of the hydrogels thus obtained ((B) according to the thesis and (A) according to the invention): the hydrogel (B) has a less homogeneous appearance than the hydrogel (A). ).

 It is also observed that these two hydrogels do not have the same behavior when they are shaken: the right-hand cliche of FIG. 13 shows these two hydrogels after shaking. Due to its thixotropic behavior, the viscosity of the hydrogel (A) according to the invention decreased during stirring and sank in the lower part of the Ependorf tube; on the other hand, the agitation did not cause flow of the hydrogel (B) which remained in the upper part of the Ependorf tube (conical end).

Example 5 - preparation of a q-lactalbumin hydrogel at pH 7 and 80 ° C.

 The purpose of this test and to reproduce the hydrogel described by Kavanagh, G.M., A.H. Clark, et al. (2000). "Heat-induced gelation of beta-lactoglobulin / alpha-lactalbumin blends at pH 3 and pH 7." Macromolecules 33 (19): 7029-7037, then characterize its rheological properties.

Conditions described in the article:

 A concentration of 15% (w / w), ie 150 mg / mL

 T ° C = 80 ° C

 Solvent = deionized water

 pH = 7.0

 The gels are observed after 1 to 2 hours at 80 ° C.

Conditions implemented:

A-La concentration of 15% (w / w), that is 150 mg / mL T ° C = 80 ° C during lh

 Solvent = deionized water

 pH = 7.2

The verification of the a-La concentration is carried out by measuring the absorbance at 280 nm of the solution using a Nanodrop® ND-1000 spectrophotometer (LabTech): the measurements are performed on three diluted protein solutions each in the fifth: Al = 61; A2 = 62; A3 = 61. The concentration of a-La is determined according to the Beer-Lambert law A = ε C 1 where ε = 27880 L-mo '-cm ^"1 and 1 = 1 cm The molar concentration C of the solution A-La is therefore 10.9 mM, which corresponds to a mass concentration of 154 mg / mL (the molar mass of α-La is 14150 g mol ^-1 ).

 The prepared a-La solution is separated into two 200 μL tubes. One tube is placed at 80 ° C, 1h, without stirring and the other tube is stored at room temperature as a control.

 In less than one hour, a hydrogel formed in the tube at 80 ° C. The gel obtained is hard, a cone does not sink in but shows some elasticity. It can also be demolded while retaining the shape of the tube which is not the case of the hydrogels according to the invention which have a softer consistency.

 The hydrogel thus obtained also has a more transparent appearance while the hydrogels according to the invention are translucent (they let a diffuse light pass but we can not distinguish objects through these hydrogels). Finally, if you shake it strongly, it does not change shape, it is irreversible.

The hydrogel produced here thus has neither the appearance nor the physical properties of the thixotropic gels made according to the process of the invention.

BIBLIOGRAPHY

 Akkermans, C., P. Venema, et al. (2008). "Peptides are building blocks of heat-induced fibrillar protein aggregates of beta-lactoglobulin formed at pH 2." Biomacromolecules 9 (5): 1474-1479.

Audic, J. L, Chaufer B, et al. (2003). "Non-food applications of milk components and dairy coproducts: A review." Milk 83 (6): 417-438.

Chen, H. (1995). "Functional properties and applications of edible films made of milk proteins."

Journal of Dairy Science 78 (11): 2563-2583.

Gosal, W.S., A.H. Clark, et al. (2004). "Fibrillar beta-lactoglobulin gels: Part 1. Fibril formation and structure." Biomacromolecules 5 (6): 2408-2419.

 Gosal, W.S., A.H. Clark, et al. (2004). "Fibrillar Beta-Lactoglobulin Gels: Part 2. Dynamic Mechanical Characterization of Heat-set Systems." Biomacromolecules 5 (6): 2420-2429.

Gosal, W.S., A.H. Clark, et al. (2004). "Fibrillar beta-lactoglobulin gels: Part 3. Dynamic mechanical solvent-induced systems." Biomacromolecules 5 (6): 2430-2438.

Graveland-Bikker, J. F., R. Ipsen, et al. (2004). "Influence of calcium on the self-assembly of partially hydrolyzed alpha-lactalbumin." Langmuir 20 (16): 6841-6846.

Gunasekaran, S., S. Ko, et al. (2007). "Use of whey proteins for encapsulation and controlled delivery applications." Journal of Food Engineering 83 (1): 31-40.

Heidebach, T., P. Forst, et al. (2009). "Microencapsulation of probiotic cells by means of rennet-gelation of milk proteins." FOOD HYDROCOLLOIDS 23 (7): 1670-1677.

 Heidebach, T., P. Forst, et al. (2009). "Transgutaminase-induced caseinate gelation for the microencapsulation of probiotic cells." INTERNATIONAL DAIRY JOURNAL 19 (2): 77-84. Hines, M. E. and E. Foegeding (1993). "INTERACTIONS OF ALPHA-LACTALBUMIN AND BOVINE SERUM-ALBUMIN WITH BETA-LACTOGLOBULIN IN THERMALLY INDUCED GELATION." Journal of Agricultural and Food Chemistry 41 (3): 341-346.

 Holmes, T.O., S. de Lacalle, et al. (2000). "Extensive neurite outgrowth and active synapse training on self-assembling peptide scaffolds." Proceedings of the National Academy of Sciences of the United States of America 97 (12): 6728-6733.

Ipsen, R. and J. Otte (2007). "Self-assembly of partially hydrolyzed alpha-lactalbumin." BIOTECHNOLOGY ADVANCES 25 (6): 602-605.

 Ipsen, R., J. Otte, et al. (2001). "Molecular self-assembly of partially hydrolyzed alpha-lactalbumin resulting in strong gels with a novel microstructure." Journal of Dairy Research 68 (2): 277-286.

 Kamau, S.M., S.C. Cheison, et al. (2010). "Alpha-Lactalbumin: Its Bioactive Production Technologies and Peptides." Comprehensive Reviews in Food Science and Food Safety 9 (2): 197-

212.

 Kavanagh, G.M., A.H. Clark, et al. (2000). "Heat-induced gelation of beta-lactoglobulin / alpha-lactalbumin blends at pH 3 and pH 7." Macromolecules 33 (19): 7029-7037.

 Kavanagh, G.M., A.H. Clark, et al. (2000). "Heat-induced gelation of globular proteins: part 3.

 Molecular studies on low beta beta-lactoglobulin gels. "International Journal of Biological

Macromolecules 28 (1): 41-50.

 Kopecek, J. and J. Yang (2009). "Peptide-directed self-assembly of hydrogels." Acta Biomater 5 (3):

 805-816.

 Kyle, S., A. Aggeli, et al. (2009). "Production of self-assembly biomaterials for tissue engineering." Trends in Biotechnology 27 (7): 423-433.

 Tien, O, C. Vachon, et al. (2001). "Milk protein coatings prevent oxidative browning of apples and potatoes." Journal of Food Science 66 (4): 512-516.

Livney, Y. D. (2010). "Milk proteins as vehicles for bioactives." Current Opinion in Colloid & Interface

Science 15 (1-2): 73-83. Loveday, SM, MA Rao, et al. (2009). "Factors Affecting the Rheological Characteristics of Fibril Gels: The Case of Beta-Lactoglobulin and Alpha-Lactalbumin." Journal of Food Science 74 (3): R47-R55.

 Madureira, A.R., C.I.Pereira, et al. (2007). "Bovine Whey Proteins - Overview on their main biological properties." Food Research International 40 (10): 1197-1211.

 Mewis, J. (1979). "THIXOTROPY - GENERAL-REVIEW." Journal of Non-Newtonian Fluid Mechanics 6 (1): 1-20.

 Oboroceanu, D., L. Wang, et al. (2010). "Characterization of β-Lactoglobulin Fibrillar Assembly Using Microscopy Atomic Force, Polyacrylamide Gel Electrophoresis, and In Situ Fourier Transform Infrared Spectroscopy." Journal of Agricultural and Food Chemistry.

 Paulsson, M., P. O. Hegg, et al. (1986). HEAT-INDUCED GELATION OF INDIVIDUAL WHEY

DYNAMIC RHEOLOGICAL STUDY PROTEINS. "Journal of Food Science 51 (1): 87-90.Pek, YS, ACA Wan, et al (2008)." A thixotropic nanocomposite gel for three-dimensional cell culture. "Nature Nanotechnology 3 (11): 671-675.

Permyakov, E.A. and L.J. Berliner (2000). "alpha-Lactalbumin: structure and function." Febs Letters

 473 (3): 269-274.

 Piau, J. M. (2007). "Carbopol gels: Elastoviscoplastic and slippery glasses made of individual swollen sponges Meso- and macroscopic properties, constitutive equations and scaling laws." Journal of Non-Newtonian Fluid Mechanics 144 (1): 1-29.

Pinion, F., A. Magnin, et al. (1998). "Thixotropic behavior of clay dispersions: Combinations of scattering and rheometric techniques." Journal of Rheology 42 (6): 1349-1373.

Restani, P., Ballabio C., et al. (2009). "Molecular aspects of milk allergens and their role in clinical events." ANALYTICAL AND BIOANAL YTICAL CHEMISTRY 395 (1): 47-56.

Rodrigues, M. MA, A. R. Simioni, et al. (2009). "Preparation, characterization and in vitro cytotoxicity of BSA-based nanospheres containing nanosized magnetic particles and / or photosensitizer." Journal of Maqnetism and Materials Materials 321 (10): 1600-1603.

Semo, E., Kesselman, E., et al. (2007). "Casein micelle as a natural nano-capsular vehicle for nutraceuticals." Food Hydrocolloids 21 (5-6): 936-942.

Song, F., L. Zhang, et al. (2009). "Genipin-crosslinked casein hydrogels for controlled drug delivery." INTERNATIONAL JOURNAL OF PHARMACEUTICS 373 (1-2): 41-47.

 Tokpavi, D. L., P. Jay, et al. (2009). "Experimental study of the very slow flow of a yield stress fluid around a circular cylinder." Journal of Non-Newtonian Fluid Mechanics 164 (1-3): 35-44. Van der Linden, E. and P. Venema (2007). "Self-assembly and aggregation of proteins." Current Opinion in Colloid & Interface Science 12 (4-5): 158-165.

Vintiloiu, A. and J.-C. Leroux (2008). "Organogels and their use in drug delivery - A review." JOURNAL OF CONTROLLED RELEASE 125 (3): 179-192.

Yan, H., H. Frielinghaus, et al. (2008). "Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway." Soft Matter 4 (6): 1313-1325.

Yan, H., Saiani, A., et al. (2006). "Thermoreversible Protein Hydrogel Cell Scaffold." Biomacromolecules 7 (10): 2776-2782.

 Yanlian, Y., K. Ulung, et al. (2009). "Designer self-assembling peptide nanomaterials." Nano Today

 4 (2): 193-210.

 Zhang, S. (2008). Designer self-assembling nanofiber peptide scaffolds for study of 3-D cell biology and beyond. Advances in Cancer Research, Vol 99. San Diego, Elsevier Academy Press Inc. 99: 335- +.

Claims

A process for preparing an α-lactalbumin hydrogel from an aqueous suspension of α-lactalbumin at a concentration C _a- of from 5 to 60 mg / mL, comprising the steps of:

 a) suspending α-lactalbumin in an acidic aqueous solution having an ionic strength less than or equal to 60 mM; said suspending consisting of:

(al) preparing an acidic aqueous solution having a proton concentration expressed in mM determined by the sum of: (numerical value of C _a- expressed in g / L) + 10;

 (a2) suspending Ρα-lactalbumin in said acidic aqueous solution; and

 (a3) if necessary, adjusting the pH to a value between 1.5 and 2.5;

 b) forming the gel from said suspension of α-lactalbumin obtained at the end of step a); said formation of the gel is carried out under the following conditions:

 - at a temperature below 60 ° C;

 under agitation having an intensity defined by a Reynolds number between 37 and 1000;

 - for 10 hours to 1 week, and

 in the absence of evaporation of water of said suspension of α-lactalbumin.

2. Method according to claim 1, characterized in that the Reynolds number is between 300 and 500.

 3. Method according to claim 1 or claim 2, characterized in that the temperature is between 35 and 55 ° C.

 4. Method according to any one of the preceding claims, characterized in that the pH is between 1.8 and 2.2.

 5. Method according to any one of the preceding claims, characterized in that said suspension of α-lactalbumin obtained at the end of step a) is filtered.

6. Rheofluidifying, threshold and thixotropic hydrogel of α-lactalbumin obtainable according to the method of claims 1 to 5.

7. Rheofluidifying, threshold and thixotropic α-lactalbumin hydrogel having an α-lactalbumin content of between 5 and 60 mg / mL, a pH of between 1.5 and 2.5 and an ionic strength less than or equal to 60 mM.

 8. Use of a thyrotropic, threshold and thixotropic hydrogel of α-lactalbumin having an α-lactalbumin content of between 5 and 60 mg / mL as a food texturizer.

 9. Food product comprising at least one a-lactalbumin threshold and thixotropic rheofluidifying hydrogel having a α-lactalbumin content of between 5 and 60 mg / ml.

 10. Rheofluidifying, threshold and thixotropic α-lactalbumin hydrogel having an α-lactalbumin content of between 5 and 60 mg / mL, for use in the treatment and / or healing of wounds.

 11. Dressing comprising at least one a-lactalbumin threshold and thixotropic rheofluidifying hydrogel having an α-lactalbumin content of between 5 and 60 mg / ml, and optionally at least one active compound such as a cicatrizing agent or an antimicrobial agent.

 12. Use of a shifted, thresh- sive and thixotropic hydrogel of α-lactalbumin having an α-lactalbumin content of between 5 and 60 mg / ml for the preparation of a cosmetic composition.

 13. Cosmetic composition comprising at least one a-lactalbumin threshold and thixotropic rheofluidifying hydrogel of α-lactalbumin having an α-lactalbumin content of between 5 and 60 mg / ml.

 14. Use of a Rheofluidifying, Threshold and Thixotropic Hydrogel of α-Lactalbumin Having an α-Lactalbumin Content of 5 to 60 mg / mL, for the Preparation of Paints.

 15. Paint comprising at least one rheofluidifying, threshold and thixotropic hydrogel of α-lactalbumin having an α-lactalbumin content of between 5 and 60 mg / ml.